Robert William Chambers was born on in Brooklyn, New York to William P. Chambers, a lawyer, and Caroline Chambers (née Boughton), a direct descendant of Roger Williams, founder of Providence, Rhode Island. Robert's brother was Walter Boughton Chambers, a progressive and influential architect. Robert dedicated his most famous book, The King in Yellow, to Walter.

Robert Chambers was first educated at the Brooklyn Polytechnic Institute, and then entered the Art Students' League at the age of twenty — where the artist Charles Dana Gibson was a fellow student. He and Gibson later created the image of the jet-set Gibson Girl, sometimes called the Chambers Girl. Chambers later studied at the École des Beaux-Arts and at Académie Julian in Paris from 1886 to 1893, and his work was displayed as early as 1889 in the Paris Salon. On his return to New York, he succeeded in selling illustrations to Life, Truth, and Vogue magazines. Then, for reasons unclear at this time, he devoted most of his time to writing, producing his first novel, In the Quarter, in 1894.

His most famous, and probably most meritorious, work was The King in Yellow, a 1895 collection of weird, fantasy, and science fiction short stories — connected by the theme of a fictitious drama, The King in Yellow, which causes those who read it to become mentally unbalanced. Genre critic E. F. Bleiler described The King in Yellow as one of the most important works of American supernatural fiction. Bleiler also wrote that Chambers was the link between the work of Poe and the modern writers of supernatural fiction.

Chambers returned to the weird genre in his later short story collections The Maker of Moons and The Tree of Heaven, but neither was as successful as The King in Yellow. Several of his most popular stories are considered to be science fiction, including “In Search of the Unknown,” “Police!!!,” and “The Repairer of Reputations.”

In order to earn a living, Chambers turned to writing romantic fiction. According to historians, Chambers was one of the most successful authors of his period, his later novels selling well — with a handful achieving best-seller status. Many of his works were also serialized in magazines. After 1924 he devoted himself solely to writing historical fiction.

Over his lifetime, Chambers had more than 70 books published, with fourteen of them turned into movies. One of his books, The Common Law, was filmed three times during his lifetime. He also wrote much verse, many short stories, articles, and two stage and opera books which were performed. Chambers always traveled with the rich and powerful. That he knew Rupert Hughes, the uncle of Howard Hughes, is a well-known fact. Rupert Hughes wrote the introduction to a 1938 edition of The King in Yellow.

Chambers for several years made his summer residence in a remodeled ancestral estate at Broadalbin in the foothills of the Adirondacks. He also maintained an office near Central Park, the location of which was a secret even from his own family. Some of his novels touch upon colonial life in Broadalbin and nearby Johnstown.

On July 12, 1898, he married Elsa Vaughn Moller (1882 - 1939). They had a son, Robert Edward Stuart Chambers (later known as Robert Husted Chambers), who also gained some fame as an author.

Robert W. Chambers died on in New York City, following abdominal surgery.

An article on the fantastic fiction of Chambers by Jon D. Swartz was published in The National Fantasy Fan for January, 2017 (Volume 76, Number 1).